Shigemi Matsuyama, Ph.D
Office/Laboratory: Wolstein Research Building 3 F.
Cancer Cell Biology, Cell Death Regulation, Cell Penetrating Peptide.
My laboratory has two research projects as explained below:
(1) How does the stressed cell decide its fate to live or die?
Several types of stresses, including DNA damage, protein misfolding, reactive oxygen
species generation, viral infection and tropic factor deprivation are known to activate
apoptosis pathways controlled by evolutionarily conserved Bcl-2 family proteins
(Reviewed in 1-3). There are two types of Bcl-2 family proteins: anti-apoptotic
and pro-apoptotic proteins. Bcl-2, Bcl-X, and Mcl-1 are anti-apoptotic proteins
that inhibit cell death through several types of stress. Bax, Bak, Bid, and Bim
are the well-known members in pro-apoptotic Bcl-2 family proteins, and these proteins
trigger apoptotic signal cascades that lead the stressed cell to undergo apoptosis.
Cellular stresses activate pro-apoptotic Bcl-2 family proteins and these pro-apoptotic
proteins initiate the release of apoptogenic factors (e.g. cytochrome c) from mitochondria.
Contrarily, anti-apoptotic Bcl-2 family proteins work as antagonists to inhibit
the release of apoptogenic factors from mitochondria. Therefore, the balance of
pro- and anti-apoptotic Bcl-2 family proteins is one important factor in deciding
whether a cell dies or survives.
One of the fundamental unanswered questions in apoptosis regulation is the mechanism
controlling how pro-apoptotic Bcl-2 family proteins (such as Bax) are activated
by stresses. In other words, how do different types of stresses activate a single
Bax molecule to induce apoptosis? Is there any specific mediator for each stress,
and/or is there any common upstream stress sensor mediating the degree of stresses
Recently, my laboratory found that Ku70 is one of the Bax-inhibiting factors that
keeps Bax inactive in the cytosol (summarized in 4, 5). Importantly, Ku70 is a ubiquitously
expressed and evolutionarily conserved protein that has been known to play an important
role in DNA double-strand break repair 6. We have found that the cytosolic Ku70
has a novel function as a Bax inhibitor, independent from Ku70’s previously
known function in DNA repair in the nucleus. We hypothesize that Ku70 is a stress
sensor protein that controls the early phase of Bax activation, and that the Ku70
modification influencing the Ku70-Bax interaction sets the sensitivity of the cells
to determine whether to initiate apoptosis.
To test our hypothesis, our research currently has the following aims:
Aim 1: To determine the mechanism of post-translational control of Ku70 levels and
functions in normal and stressed cells.
Our laboratory has found that ubiquitin-dependent modification of Ku70 is one of
the mechanisms to decrease Ku70 levels in apoptotic cells 7. In addition, the acetylation
of Ku70 has also been shown to induce the dissociation of Ku70 from Bax by other
groups 8, 9. We are currently studying the impact of these post-translational modifications
on the half-life, subcellular localization, affinities to binding proteins (Bax
and Ku80) and biological activities which regulate cell death and DNA repair. We
are also searching for the Ku70 modifying enzymes that mediate cellular stresses
to this sensor protein.
Aim 2: To determine the molecular mechanism behind the Ku70-dependent inhibition
of Bax activation.
We are currently studying how the binding of Ku70 inhibits the activation process
of Bax, such as the conformational change of Bax and the interaction with Bax activators
such as BH3 only proteins that are the members of pro-apoptotic Bcl-2 family proteins.
Aim 3: To determine the biological significance of Ku70-Bax interaction to control
cell death sensitivity using mouse genetics.
Ku70-/- mice have been generated by other groups 10, 11 and these mutant mice show
the phenotypes of growth retardation, accelerated replicative senescence of cultured
fibroblasts, increased cell death in gastrointestinal tissue and T-cell lymphoma
development. We hypothesize that the disease phenotypes of Ku70-/- mice may be,
at least in part, due to the increased Bax-mediated cell death caused by the absence
of Ku70. Bax-/- mice have been established by other group 12. By crossing Ku70-deficient
and Bax-deficient mice, we are generating Ku70-/-Bax-/- mice and will analyze the
phenotype of the Ku70-/-Bax-/- mice to determine whether Bax deletion corrects some
of the disease phenotypes of a Ku70-/- mouse.
(2) Development of Cytoprotective Cell-Penetrating Penta-Peptides
The cellular membrane has a strict selectivity for molecular transport to maintain
cellular life. Because of this selectivity, delivering potentially effective drugs
into damaged cells often become difficult. Therefore, there is a strong need to
develop the technologies for molecular transport across the cellular membrane. My
laboratory has discovered specific penta-peptides (5-amino-acid peptides) that penetrate
the cellular membrane (please see the next paragraph for details) 4, 5. These penta-peptides
are categorized as the shortest cell-penetrating peptides13, and the mechanism of
how these peptides penetrate the cellular membrane is now being investigated in
my laboratory. By elucidating the cell penetrating mechanism of these penta-peptides,
we hope to develop the technology to deliver effective drugs into the cell.
Development of Bax-Inhibiting Peptide
Previously, we found that Ku70 binds Bax and inhibits Bax-mediated cell death 7.
We identified the Bax binding domain of Ku70 and the penta-peptide derived from
this domain was sufficient to bind and inhibit Bax4. Interestingly, these penta-peptides
entered the cells when the peptides were added to the culture medium, indicating
that these peptides are cell permeable. These peptides have been named Bax-Inhibiting
Peptides (BIPs). The discovery of BIPs brought us two different types of research
opportunities: (a) Utilization of BIPs to rescue damaged cells, and (b) Elucidation
of the mechanism of membrane penetration of BIPs.
(a) Utilization of BIPs to rescue damaged cells
The BIP has been shown to suppress drug-induced apoptosis in cell culture 4, 14.
Importantly, the BIP rescued retinal ganglion cells from optic nerve injury-induced
apoptosis and also increased the survival rate of the hepatocytes after transplantation
in mice15, 16. Currently, we are examining whether BIP can be utilized to protect
damaged tissue by ischemia-reperfusion treatment in a mouse model. We are also developing
more effective cytoprotective, cell-permeable peptides by introducing mutations
(b) Elucidation of the mechanism of membrane penetration of BIPs and BIP-derived
In addition to BIPs, we have also developed new cell-permeable peptides by mutating
the sequence of BIPs. These mutant peptides do not bind Bax and thus do not inhibit
cell death. BIP and these mutant peptides have been labeled “BIP-derived
Cell Penetrating Penta-Peptides (BCP)” 5.
BCPs belong to the growing family called “Cell Penetrating Peptides”
that include the Human Immunodeficiency Virus (HIV) tat peptide13. The cell entry
mechanism of cell-penetrating peptides is not yet known, and it is possible that
there are different mechanisms depending on the type of cell-penetrating peptide.
My laboratory is focusing on the cell entry mechanism of BCPs. We are currently
examining the following questions: (1) Do BCPs use energy-dependent and/or energy-independent
mechanism(s) to enter the cell? (2) Is there any unidentified receptor molecule(s)
on the cell surface that is required for the cell entry of BCPs? (3) Can we design
new penta-peptides that have improved cell entry activity? Our study will advance
the understanding of the protein traffic in the plasma membrane, and we hope that
our study will contribute the development of new drug delivery technology into the
1. Reed, J. C. Double identity for
proteins of the Bcl-2 family. Nature 387, 773-6. (1997).
2. Korsmeyer, S. J. BCL-2 gene family
and the regulation of programmed cell death. Cancer Res 59, 1693s-1700s. (1999).
3. Green, D. R. At the gates of
death. Cancer Cell 9, 328-30 (2006).
4. Yoshida, T. et al. Bax-inhibiting
peptide derived from mouse and rat Ku70. Biochem Biophys Res Commun 321, 961-6 (2004).
5. Gomez, J. A., Gama, V., Matsuyama,
S. Cell-permeable penta-peptides derived from Bax-inhibiting peptide. Cell Penetrating
Peptide, 2nd edition., 469-481 (2006).
6. Downs, J. A. & Jackson, S.
P. A means to a DNA end: the many roles of Ku. Nat Rev Mol Cell Biol 5, 367-78 (2004).
7. Gama, V. et al. Involvement of
the ubiquitin pathway in decreasing Ku70 levels in response to drug-induced apoptosis.
Exp Cell Res 312, 488-99 (2006).
8. Cohen, H. Y. et al. Acetylation
of the C terminus of Ku70 by CBP and PCAF controls Bax-mediated apoptosis. Mol Cell
13, 627-38 (2004).
9. Subramanian, C., Opipari, A.
W., Jr., Bian, X., Castle, V. P. & Kwok, R. P. Ku70 acetylation mediates neuroblastoma
cell death induced by histone deacetylase inhibitors. Proc Natl Acad Sci U S A 102,
10. Gu, Y. et al. Growth retardation and leaky
SCID phenotype of Ku70-deficient mice. Immunity 7, 653-65 (1997).
11. Li, G. C. et al. Ku70: a candidate tumor
suppressor gene for murine T cell lymphoma. Mol Cell 2, 1-8 (1998).
12. Knudson, C. M., Tung, K. S., Tourtellotte,
W. G., Brown, G. A. & Korsmeyer, S. J. Bax-deficient mice with lymphoid hyperplasia
and male germ cell death. Science 270, 96-9. (1995).
13. Joliot, A. & Prochiantz, A. Transduction
peptides: from technology to physiology. Nat Cell Biol 6, 189-96 (2004).
14. Zi, X. & Simoneau, A. R. Flavokawain
A, a novel chalcone from kava extract, induces apoptosis in bladder cancer cells
by involvement of Bax protein-dependent and mitochondria-dependent apoptotic pathway
and suppresses tumor growth in mice. Cancer Res 65, 3479-86 (2005).
15. Qin, Q., Patil, K. & Sharma, S. C. The
role of Bax-inhibiting peptide in retinal ganglion cell apoptosis after optic nerve
transection. Neurosci Lett 372, 17-21 (2004).
16. Tanaka, K. et al. Prolonged survival of
mice with acute liver failure with transplantation of monkey hepatocytes cultured
with an antiapoptotic pentapeptide V5. Transplantation 81, 427-37 (2006).
Publication by Shigemi Matsuyama (1997-) and Matsuyama laboratory (from 2003-).
1. Shendel SL, Xie Z, Montal MO, Matsuyama S, Montal M,
Reed JC (1997) Channel formation by anti-apoptotic protein, Bcl-2.
Proc Natl Acad Sci USA 94, 5113-5118.
2. Matsuyama S, Xu Q, Velrous J, Reed JC (1998) F0F1-proton-pump
ATPase is required for the function of a pro-apoptotic protein, Bax, both in yeast
and mammalian cells. Molecular Cell 1: 327-336.
3. Xie Z, Schendel SL, Matsuyama S, Reed JC (1998) Acidic pH
promotes dimerization of Bcl-2 family proteins. Biochemistry
4. Matsuyama S (1998) Molecular mechanism of Bax-induced cell
death. Cell Technology 17:891-898 (Japanese article).
5. Xu Q, Jurgensmeier JM, Zha H, Matsuyama S, Reed JC (1998)
Exploiting yeast for the investigation of mammalian proteins that regulate programmed
cell death. Apoptosis Detection and Assay Methods, Edited by Li Zhu and Jerald Chun,
BioTechniques Books, Natick, MA. Page 93-115.
6. Reed JC, Jurgensmeir J, Matsuyama S (1998) Bcl-2 family
proteins and mitochondria (Review). Biochemica et Biophisica Acta,
7. Marzo I, Brenner C, Zamzami N, Jurgensmeier JM, Susin SA, Vieira
HLA, Prevost MC, Xie Z, Matsuyama S, Reed JC, Kroemer G. (1998) Bax and adenine
nucleotide translocator cooporate in the mitochondrial control of apoptosis.
Science, 281. 2027-2031.
8. Matsuyama S, Schendel SL, Xie Z, Reed JC (1998) Cytoprotection
by Bcl-2 requires the pore-forming a5 and a6 helicies. Journal of Biological
Chemistry 273. 30995-31001.
9. Matsuyama S (1999) Mitochondria dependent cell death
pathway. In "Apoptosis and the digestive organ-diseases" (Edited by H.
Ishi), p.p. 31-46 (Japanese article)
10. Matsuyama S, Nouraini S, Reed JC (1999) Yeast as a tool for apoptosis
research. Current Opinion in Microbiology 2:618-623.
11. Xu Q, Matsuyama S , Reed JC (2000) Utilization of yeast genetics
to explore mammalian cell death mechanism. Methods in Enzymology
12. Matsuyama S (1999) Advantages of the utilization of yeast genetics
to study apoptosis mechanism. Experimental Medicine (Japanese article). 17, 2607-2608.
13. Matsuyama S, Llipos J, Devraux Q, Tsien R, Reed JC (2000) Changes in
intramitochondrial and cytosolic pH:early events that modulate caspase activation
during apoptosis. Nature Cell Biology, 2, 318-325.
14. Nuoraini S, Six E, Matsuyama S, Krajewski S, Reed JC (2000). The
putative pore forming-domain of Bax regulates mitochondrial localization and interaction
with Bcl-XL. Mol Cell Biol, 20, 1604-1615.
15. Zhang H, Huang Q, Matsuyama S, Ke N, Godzik A, and Reed JC (2000).
Drosophila Pro-Apoptotic Bcl-2/Bax Homologue Reveals Evolutionary Conservation
of Cell Death Mechanisms.
J Biol Chem, 275:27303-6.
16. Matsuyama S and Reed JC (2000). Mitochondria-dependent Apoptosis
and Cellular pH Regulation. (Review article) Cell Death and Differentiation,
17. Gu C, Sun W, Sawada M, Matsuyama S, Newman P (2003). PECAM-1 suppresses
Bax-mediated apoptosis. Blood, 102, 169-179.
18. Yoshida T, Sawada M, Okuno M, Gama V, Hayes P, Matsuyama S (2004). New
Bax-inhibiting peptides (BIPs) derived from mouse and rat Ku70. Biochemical
and Biophysical Research Communications, 321, 961-966.
19. Gohil V, Hayes P, Matsuyama S, Schägger H, Schlame M, Greenberg
M (2004). Cardiolipin biosynthesis and mitochondrial respiratory chain function
are interdependent. Journal of Biological Chemistry, 279:
20. Martinez JJ, Seveau S, Veiga-Chacon E, Matsuyama S, Cossart P (2005).
Identification of Ku70, a component of the DNA-dependent protein kinase, as
a receptor involved in Rickettsia conorii invasion of mammalian cells. Cell,
21. Gama V, Yoshida T, Gomez J, Mayo L, Haas A, Matsuyama S.
(2006) Involvement of ubiquitin-pathway in Ku70 decrease in response to apoptosis.
Experimental Cell Research, 312: 488-99.
22. Gomez J, Gama V, Matsuyama S. Cell permeable pentapeptides derived from
Bax inhibiting domain of Ku70 (2006). “Cell Permeable Peptides”
(2nd edition, edited by Ulo Langel), CRC Press, ,p.p. 469-481.
23. Sun W, Hattori N, Matsuyama S,hiota K (2006), “Proliferation related
acidic leucine rich protein PAL31 functions as a caspase-3 inhibitor”
Biochemical and Biophysical Research Communications, 342(3):817-23.
24. Chitamber C, Wereley JP, Kotamraju S, Matsuyama S. (2006) Induction of
Apoptosis by Galium Nitrate in Lymphoma Cells: Involvement of Bax, mitochondria,
and Proteasome Pathway. Molecular Cancer Therapeuitic,
25. Bergom C, Gao C, Matsuyama S, Newman PJ. (2006) The Cell Surface Molecule
PECAM-1 Mediates Resistance Against Chemotherapy-Induced Apoptosis., Cancer
Biology and Therapy 5: 1699-707
26. Chen YN, Yamada H, Mao W, Matsuyama S, Aihara M, Araie M. (2007).
Hypoxia-Induced Retinal Ganglion Cell Death and the Neuroprotective
Effects of Beta-adrenergic Antagonists. Brain Research,
Li Y, Ykota T, Yoshida T, Gama V, Sawada M, Ishikawa K, Cohen HY, Sinclair DA, Mizushima
H, Matsuyama S. Bax-inhibiting Peptide (BIP) suppresses poly-glutamine-induced
cell death that induces Ku70 acetylation. submitted.
Gomez J, Yoshida T, Stapleton M, Gama V, Paddock C, Newman P, Wilcox D, Matsuyama
S. Bax inhibiting peptides derived from Ku70 suppress chemotherapy-induced
cell death of megakaryocytes. submitted.